It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of Staphylococcus aureus infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the MurC embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
The enzyme UDP-N-acetylmuramate:L-alanine ligase, encoded by the gene MurC catalyses the addition of the first amino acid (L-alanine) of the peptide moiety in peptidoglycan biosynthesis. L-alanine is added to UDP-N-acetyl muramate with the concomittant hydrolysis of ATP to yield UDP-N-acetylmuramyl-L-alanine, ADP and phosphate. The gene has been cloned and sequenced from Escherichia coli and the corresponding protein has been over-expressed, purified and kinetically characterised (Liger, D., Masson, A., Blanot, D., van Heijenoort, J. & Parquet, C., Eur. J. Biochem. 230, 80-87). The kinetic mechanism of this enzyme has been investigated. (Falk, P. J., Ervin, K. M., Volk, K. S. & Ho, H. T. (1996) Biochemistry, 35, 1417-1422). The gene sequences of MurC from Bacillus subtilis and Haemophilus influenzae are also known.
The discovery of a MurC homologue in the human pathogen Staphylococcus aureus will allow us to produce UDP-N-acetylmuramate:L-alanine ligase enzyme which can then be used to screen for novel antibiotics. Inhibitors of this protein have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known MurC protein.